Ceramide anionic liposome compositions

ABSTRACT

Described herein are pharmaceutical compositions according to aspects of the present invention which include one or more hydrophilic antineoplastic chemotherapeutics, such as vinca alkyloid antineoplastic chemotherapeutics, encapsulated in ceramide anionic liposomes. Methods of treatment of a subject having cancer using the pharmaceutical compositions are described, along with methods of making ceramide anionic liposomes which encapsulate one or more hydrophilic antineoplastic chemotherapeutics in the aqueous interior of the ceramide anionic liposomes.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/468,578, filed May 10, 2012, which claims priority from U.S.Provisional Patent Application Ser. No. 61/484,496, filed May 10, 2011,the entire content of both of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to liposome compositions fordelivery of antineoplastic chemotherapeutics to treat cancer. Aspects ofthe present invention relate to ceramide anionic liposome compositionscontaining hydrophilic antineoplastic chemotherapeutics and/or vincaalkyloids.

BACKGROUND OF THE INVENTION

There is a continuing need for anti-cancer compositions and methods oftreatment. Methods of encapsulating hydrophilic antineoplastictherapeutics are required.

SUMMARY OF THE INVENTION

Pharmaceutical compositions are provided according to aspects of thepresent invention which include a hydrophilic antineoplasticchemotherapeutic encapsulated in ceramide anionic liposomes.

According to aspects of the present invention the hydrophilicantineoplastic chemotherapeutic is a vinca alkyloid antineoplasticchemotherapeutic encapsulated in ceramide anionic liposomes.

According to aspects of the present invention the hydrophilicantineoplastic chemotherapeutic is vinblastine, vincristine,vinglycinate, vinorelbine, vindesine, sorafenib, cladribine and acombination of any two or more thereof encapsulated in ceramide anionicliposomes.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include one or more hydrophilic antineoplasticchemotherapeutics encapsulated in ceramide anionic liposomes wherein theamount of the drug encapsulated in the liposomes compared to the amountof total lipids in the liposomes is in the range of about 1:1-1:100.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include one or more hydrophilic antineoplasticchemotherapeutics encapsulated in ceramide anionic liposomes wherein theceramide anionic liposomes comprise a 3-5.5:1-4:1:3 ratio of neutrallipid:pegylated neutral lipid:anionic lipid:one or more of C4 ceramide,C6 ceramide, C8 ceramide, C10 ceramide, C12 ceramide, C14 ceramide, C16ceramide and C18 ceramide.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include one or more hydrophilic antineoplasticchemotherapeutics encapsulated in ceramide anionic liposomes wherein theceramide anionic liposomes comprise a 3-5.5:1-4:1:3 ratio of neutrallipid:pegylated neutral lipid:anionic lipid:one or more of C4 ceramide,C6 ceramide, C8 ceramide, C10 ceramide, C12 ceramide, C14 ceramide, C16ceramide and C18 ceramide, and wherein the neutral lipid is a mixture ofDSPC and DOPE, wherein the modified neutral lipid is a mixture ofPEG(2000)-DSPE andN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750, andwherein the anionic lipid is dihexadecyl phosphate.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include vinblastine encapsulated in ceramideanionic liposomes wherein the ceramide anionic liposomes comprise a3-5.5:1-4:1:3 ratio of neutral lipid:pegylated neutral lipid:anioniclipid:one or more of C4 ceramide, C6 ceramide, C8 ceramide, C10ceramide, C12 ceramide, C14 ceramide, C16 ceramide and C18 ceramide, andwherein the neutral lipid is a mixture of DSPC and DOPE, wherein themodified neutral lipid is a mixture of PEG(2000)-DSPE andN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750, andwherein the anionic lipid is dihexadecyl phosphate.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include vinblastine, vincristine, vinglycinate,vinorelbine, vindesine, sorafenib, cladribine, or a combination of anytwo or more thereof, encapsulated in ceramide anionic liposomes whereinthe ceramide anionic liposomes comprise a 3-5.5:1-4:1:3 ratio of neutrallipid:pegylated neutral lipid:anionic lipid:one or more of C4 ceramide,C6 ceramide, C8 ceramide, C10 ceramide, C12 ceramide, C14 ceramide, C16ceramide and C18 ceramide, and wherein the neutral lipid is a mixture ofDSPC and DOPE, wherein the modified neutral lipid is a mixture ofPEG(2000)-DSPE andN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750, andwherein the anionic lipid is dihexadecyl phosphate.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include one or more hydrophilic antineoplasticchemotherapeutics encapsulated in ceramide anionic liposomes wherein theliposomes comprise at least one pegylated neutral lipid, wherein thetotal amount of pegylated neutral lipid is an amount in the range of5-20 Molar percent, inclusive, and wherein at least half of the amountof included modified neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750; atleast one anionic lipid, wherein the total amount of anionic lipid is anamount in the range of 5-15 Molar percent, inclusive; a ceramideselected from C4 ceramide, C6 ceramide, C8 ceramide, C10 ceramide, C12ceramide, C14 ceramide, C16 ceramide and C18 ceramide, or a combinationof any two or more thereof, in an amount in the range of 1-40 Molarpercent, inclusive; and cationic or neutral lipids, with the provisothat the resulting ceramide anionic liposomes have a net negative chargeat physiological pH.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include one or more hydrophilic antineoplasticchemotherapeutics encapsulated in ceramide anionic liposomes wherein theliposomes comprise at least one pegylated neutral lipid, wherein thetotal amount of pegylated neutral lipid is an amount in the range of5-20 Molar percent, inclusive, and wherein at least half of the amountof included modified neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of 5-15 Molar percent,inclusive; C6 ceramide in an amount in the range of 1-40 Molar percent,inclusive; and cationic or neutral lipids, with the proviso that theresulting ceramide anionic liposomes have a net negative charge atphysiological pH.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include one or more hydrophilic antineoplasticchemotherapeutics encapsulated in ceramide anionic liposomes wherein theliposomes comprise at least one pegylated neutral lipid, wherein thetotal amount of pegylated neutral lipid is an amount in the range of5-20 Molar percent, inclusive, and wherein at least half of the amountof included modified neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of 5-15 Molar percent,inclusive; C8 ceramide in an amount in the range of 1-40 Molar percent,inclusive; and cationic or neutral lipids, with the proviso that theresulting ceramide anionic liposomes have a net negative charge atphysiological pH.

Pharmaceutical compositions are provided according to aspects of thepresent invention which include one or more hydrophilic antineoplasticchemotherapeutics encapsulated in ceramide anionic liposomes wherein theliposomes comprise at least one pegylated neutral lipid, wherein thetotal amount of pegylated neutral lipid is an amount in the range of5-20 Molar percent, inclusive, and wherein at least half of the amountof included modified neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of 5-15 Molar percent,inclusive; C6 ceramide and C8 ceramide in an amount in the range of 1-40Molar percent, inclusive; and cationic or neutral lipids, with theproviso that the resulting ceramide anionic liposomes have a netnegative charge at physiological pH.

Methods of treatment of a subject in need thereof are provided accordingto aspects of the present invention which include administration of atherapeutically effective amount of a pharmaceutical compositionaccording to aspects of the present invention which includes ahydrophilic antineoplastic chemotherapeutic encapsulated in ceramideanionic liposomes.

Methods of treatment of a subject having, or at risk of having, cancerare provided according to aspects of the present invention which includeadministration of a therapeutically effective amount of a pharmaceuticalcomposition according to aspects of the present invention which includesa hydrophilic antineoplastic chemotherapeutic encapsulated in ceramideanionic liposomes.

Methods of treatment of a subject having, or at risk of having, cancerare provided according to aspects of the present invention which includeadministration of a therapeutically effective amount of a pharmaceuticalcomposition according to aspects of the present invention which includesa hydrophilic antineoplastic chemotherapeutic encapsulated in ceramideanionic liposomes and further include administration of an adjunctanti-cancer treatment.

Methods of producing a pharmaceutical composition including ahydrophilic antineoplastic chemotherapeutic encapsulated in ceramideanionic liposomes are provided according to aspects of the presentinvention which include providing a lipid mixture comprising at leastone pegylated neutral lipid, wherein the total amount of pegylatedneutral lipid is an amount in the range of 5-20 Molar percent,inclusive, and wherein at least half of the amount of included pegylatedneutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750; atleast one anionic lipid, wherein the total amount of anionic lipid is anamount in the range of 5-15 Molar percent, inclusive; C4 ceramide, C6ceramide, C8 ceramide, C10 ceramide, C12 ceramide, C14 ceramide, C16ceramide, C18 ceramide, or a combination of any two or more thereof, inan amount in the range of 1-40 Molar percent, inclusive; and cationic orneutral lipids, with the proviso that the resulting lipid mixture has anet negative charge at physiological pH; sonicating the lipid mixture inthe presence of an amount of a antineoplastic chemotherapeutic at atemperature in the range of 55-75 degrees Celsius, inclusive, to producea sonicated mixture; and passing the sonicated mixture through a filterhaving pores of a desired size to produce liposomes having the desiredsize, at a temperature in the range of 67-75 degrees Celsius, producinga population of ceramide anionic liposomes, wherein the populationcomprises greater than 10%, greater than 25%, greater than 50% or in therange of about 15-75% of the amount of the antineoplasticchemotherapeutic, wherein the amount of the drug encapsulated in theliposomes compared to the amount of total lipids in the liposomes is inthe range of about 1:1-1:100.

Methods of producing a pharmaceutical composition including ahydrophilic antineoplastic chemotherapeutic encapsulated in ceramideanionic liposomes are provided according to aspects of the presentinvention which include providing a lipid mixture comprising at leastone pegylated neutral lipid, wherein the total amount of pegylatedneutral lipid is an amount in the range of 5-20 Molar percent,inclusive, and wherein at least half of the amount of included pegylatedneutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750; atleast one anionic lipid, wherein the total amount of anionic lipid is anamount in the range of 5-15 Molar percent, inclusive; C6 ceramide, C8ceramide, or a combination thereof, in an amount in the range of 1-40Molar percent, inclusive; and cationic or neutral lipids, with theproviso that the resulting lipid mixture has a net negative charge atphysiological pH; sonicating the lipid mixture in the presence of anamount of a antineoplastic chemotherapeutic at a temperature in therange of 55-75 degrees Celsius, inclusive, to produce a sonicatedmixture; and passing the sonicated mixture through a filter having poresof a desired size to produce liposomes having the desired size, at atemperature in the range of 67-75 degrees Celsius, producing apopulation of ceramide anionic liposomes, wherein the populationcomprises greater than 10%, greater than 25%, greater than 50% or in therange of about 15-75% of the amount of the antineoplasticchemotherapeutic, wherein the amount of the drug encapsulated in theliposomes compared to the amount of total lipids in the liposomes is inthe range of about 1:1-1:100.

According to aspects of methods of producing a pharmaceuticalcomposition including a hydrophilic antineoplastic chemotherapeuticencapsulated in ceramide anionic liposomes of the present invention, theantineoplastic chemotherapeutic is a vinca alkyloid antineoplasticchemotherapeutic.

According to aspects of methods of producing a pharmaceuticalcomposition including a hydrophilic antineoplastic chemotherapeuticencapsulated in ceramide anionic liposomes of the present invention, theantineoplastic chemotherapeutic is selected from the group consistingof: vinblastine, vincristine, vinglycinate, vinorelbine, vindesine,sorafenib, cladribine and a combination of any two or more thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing results of dynamic light scattering analysisindicating stability of ceramide anionic liposome compositions of thepresent invention;

FIG. 2 is a graph showing effects of ceramide anionic liposomecompositions of the present invention including vinblastine in a tumormodel;

FIG. 3A is a graph showing that vinblastine potentiates the inhibitoryactivity of ceramide anionic liposome compositions of the presentinvention including vinblastine on growth and survival of primary humanacute myeloid leukemia cells of poor prognosis in semisolid media forclonogeneic growth;

FIG. 3B is a graph showing in vivo activity of ceramide anionic liposomecompositions of the present invention including vinblastine on bloodburden of human acute myeloid leukemia cells in Nod Scid Gamma (NSG)mice; and

FIG. 3C is a graph showing in vivo activity of ceramide anionic liposomecompositions of the present invention including vinblastine on bonemarrow burden of human acute myeloid leukemia cells in Nod Scid Gamma(NSG) mice.

DETAILED DESCRIPTION OF THE INVENTION

Scientific and technical terms used herein are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found defined and used in context in various standardreferences illustratively including J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; B. Alberts et al., MolecularBiology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox,Lelminger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company,2004; Engelke, D. R., RNA Interference (RNAi): Nuts and Bolts of RNAiTechnology, DNA Press LLC, Eagleville, Pa., 2003; Herdewijn, P. (Ed.),Oligonucleotide Synthesis: Methods and Applications, Methods inMolecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K.Vintersten, R. Behringer, Manipulating the Mouse Embryo: A LaboratoryManual, 3rd edition, Cold Spring Harbor Laboratory Press; Dec. 15, 2002,ISBN-10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methodsand protocols in Methods Mol Biol. 2002; 185, Humana Press; CurrentProtocols in Stem Cell Biology, ISBN: 9780470151808.

The singular terms “a,” “an,” and “the” are not intended to be limitingand include plural referents unless explicitly state or the contextclearly indicates otherwise.

Ceramide anionic liposome compositions containing at least onehydrophilic antineoplastic chemotherapeutic and methods for use thereofare provided according to the present invention.

The term “hydrophilic” is well-known in the art and refers to anantineoplastic chemotherapeutic that readily absorbs water and/orreadily dissolves in water

Ceramide anionic liposome compositions containing at least one vincaalkyloid antineoplastic chemotherapeutic and methods for use thereof areprovided according to the present invention.

Vinca alkyloid antineoplastic chemotherapeutics are well-known in theart, see for example Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins, 21^(st) ed., 2005, p. 437. Vinca alkyloidantineoplastic chemotherapeutics include, but are not limited to,vinblastine, vincristine, vinglycinate, vinorelbine and vindesine.

The term “liposome” refers to a bilayer particle of amphipathic lipidmolecules enclosing an aqueous interior space. Liposomes are typicallyproduced as small unilammellar vesicles (SUVs), large unilammellarvesicles (LUVs) or multilammellar vesicles (MLVs).

An antineoplastic chemotherapeutic is associated with liposomes byencapsulation in the aqueous interior space of the liposomes, disposedin the lipid bilayer of the liposomes and/or associated with theliposomes by binding, such as ionic binding or association by van derWaals forces. Liposomes according to aspects of the invention aregenerally in the range of about 1 nanometer-1 micron in diameteralthough they are not limited with regard to size.

Size of liposomes produced according to methods of the present inventioncan be controlled using well-known techniques, including, but notlimited to, filtration through a filter having a defined pore size,extrusion and combinations thereof.

Ceramide anionic liposomes according to aspects of the present inventioninclude C4 ceramide, C6 ceramide, C8 ceramide, C10 ceramide, C12ceramide, C14 ceramide, C16 ceramide, C18 ceramide, or a combination ofany two or more thereof, one or more types of neutral or cationic lipidand at least one type of anionic lipid, such that the ceramide anionicliposomes have a net negative charge at physiological pH. Preferably, aPEG-modified lipid (pegylated lipid) is included.

According to aspects of the present invention, C6 ceramide, C8 ceramideor both C6 ceramide and C8 ceramide are included in ceramide anionicliposomes of the present invention

Ceramide anionic liposomes according to aspects of the present inventioninclude, C6 ceramide (N-Hexanoyl-D-erythro-sphingosine), one or moretypes of neutral or cationic lipid and at least one type of anioniclipid, such that the ceramide anionic liposomes have a net negativecharge at physiological pH. Preferably, a PEG-modified lipid (pegylatedlipid) is included.

Ceramide anionic liposomes according to aspects of the present inventioninclude, C8 ceramide (N-Octanoyl-D-erythro-Sphingosine), one or moretypes of neutral or cationic lipid and at least one type of anioniclipid, such that the ceramide anionic liposomes have a net negativecharge at physiological pH. Preferably, a PEG-modified lipid (pegylatedlipid) is included.

The term “cationic lipid” refers to any lipid which has a net positivecharge at physiological pH. Examples of cationic lipids include, but arenot limited to, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride (DOTMA); 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP);1,2-dioleoyl-3-dimethylammonium-propane (DODAP);dioctadecylamidoglycylspermine (DOGS);1,2-dipalmitoylphosphatidylethanolamidospermine (DPPES);2,3-dioleyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA); dimyristoyltrimethylammonium propane (DMTAP);(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium (DMRIE);dioctadecyldimethylammonium chloride (DODAC), Dimethyldidodecylammoniumbromide (DDAB); 3β[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-Chol);1-[2-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)-heptadecenyl)-3-(2-hydroxyethyl)-imidazolinium(DOTIM); bis-guanidinium-spermidine-cholesterol (BGTC);bis-guanidinium-tren-cholesterol (BGTC);1,3-Di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER)N-[3-[2-(1,3-dioleoyloxy)propoxy-carbonyl]propyl]-N,N,N-trimethylammoniumiodide (YKS-220); as well as pharmaceutically acceptable salts andmixtures thereof. Additional examples of cationic lipids are describedin Lasic and Papahadjopoulos, Medical Applications of Liposomes,Elsevier, 1998; U.S. Pat. Nos. 4,897,355; 5,208,036; 5,264,618;5,279,833; 5,283,185; 5,334,761; 5,459,127; 5,736,392; 5,753,613;5,785,992; 6,376,248; 6,586,410; 6,733,777; and 7,145,039.

The term “neutral lipid” refers to any lipid which has no net charge,either uncharged or in neutral charge zwitterionic form, atphysiological pH. Examples of neutral lipids include, but are notlimited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylethanolamine (DOPE),distearoylphosphatidylethanolamine (DSPE);1,2-dioleoyl-sn-glycero-3-Phosphocholine (DOPC), cephalin, ceramide,cerebrosides, cholesterol, diacylglycerols, and sphingomyelin.

The term “anionic lipid” refers to any lipid which has a net negativecharge at physiological pH. Examples of anionic lipids include, but arenot limited to, dihexadecylphosphate (DhP), phosphatidyl inositols,phosphatidyl serines, such as dimyristoyl phosphatidyl serine, anddipalmitoyl phosphatidyl serine, phosphatidyl glycerols, such asdimyristoylphosphatidyl glycerol, dioleoylphosphatidyl glycerol,dilauryloylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol,distearyloylphosphatidyl glycerol, phosphatidic acids, such asdimyristoyl phosphatic acid and dipalmitoyl phosphatic acid anddiphosphatidyl glycerol.

The total amount of anionic lipid included in ceramide anionic liposomesaccording to aspects of the present invention is an amount in the rangeof about 5-15 Molar percent, inclusive, more preferably an amount in therange of about 8-12 Molar percent, inclusive, still more preferably anamount in the range of about 9-11 Molar percent, inclusive, yet morepreferably about 10 Molar percent.

The term “modified lipid” refers to lipids modified to aid in, forexample, inhibiting aggregation and/or precipitation, inhibiting immuneresponse and/or improving half-life in circulation in vivo. According toaspects of the present invention, the modified lipids are neutrallipids.

Modified neutral lipids include, but are not limited to, pegylatedlipids, such as polyethyleneglycol 2000distearoylphosphatidylethanolamine (PEG(2000) DSPE); andN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(abbreviated as PEG(750) C8 herein).

The total amount of pegylated neutral lipids included in ceramideanionic liposome compositions according to aspects of the presentinvention is an amount in the range of about 5-20 Molar percent,inclusive, more preferably in the range of about 8-12 Molar percent,inclusive, still more preferably in the range of about 9-11 Molarpercent, inclusive, and yet more preferably about 10 Molar percent.

According to highly preferred aspects,N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8) is included in ceramide anionic liposome compositions ofthe present invention.

N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8) is included in ceramide anionic liposome compositionsaccording to aspects of the present invention. The total amount ofN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8) included in ceramide anionic liposome compositionsaccording to aspects of the present invention is an amount in the rangeof about 5-20 Molar percent, inclusive, more preferably in the range ofabout 8-12 Molar percent, still more preferably in the range of about9-11 Molar percent and yet more preferably about 10 Molar percent.

Two or more pegylated neutral lipids are included in ceramide anionicliposome compositions according to aspects of the present invention,wherein at least half of the amount of included total pegylated lipidsis N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8).

Pegylated neutral lipids polyethyleneglycol 2000distearoylphosphatidylethanolamine (PEG(2000) DSPE); andN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8) are included in ceramide anionic liposome compositionsaccording to aspects of the present invention. The total amount ofpolyethyleneglycol 2000 distearoylphosphatidylethanolamine (PEG(2000)DSPE) andN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8) included in ceramide anionic liposome compositionsaccording to aspects of the present invention is an amount in the rangeof about 5-20 Molar percent, inclusive, more preferably in the range ofabout 8-12 Molar percent, inclusive, still more preferably in the rangeof about 9-11 Molar percent, inclusive, and yet more preferably about 10Molar percent.

C6 ceramide is included in ceramide anionic liposome compositionsaccording to aspects of the present invention is an amount in the rangeof about 1-40 Molar percent, inclusive, more preferably in the range ofabout 5-38 Molar percent, inclusive, still more preferably in the rangeof about 10-35 Molar percent, inclusive. According to aspects of thepresent invention, C6 ceramide is included in ceramide anionic liposomecompositions is an amount of about 30 Molar percent. The C6 ceramide isnot pegylated.

C8 ceramide is included in ceramide anionic liposome compositionsaccording to aspects of the present invention is an amount in the rangeof about 1-40 Molar percent, inclusive, more preferably in the range ofabout 5-38 Molar percent, inclusive, still more preferably in the rangeof about 10-35 Molar percent, inclusive. According to aspects of thepresent invention, C8 ceramide is included in ceramide anionic liposomecompositions is an amount of about 30 Molar percent. Thus, C8 ceramide(N-Octanoyl-D-erythro-Sphingosine), which has anti-cancer activity, andpolyethyleneglycol 750 C8 ceramide(N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750, alsocalled PEG(750) C8 herein), which has substantially no anti-canceractivity compared to N-Octanoyl-D-erythro-Sphingosine, are both includedin liposomes according to aspects of the present invention,

According to aspects of the present invention, the amount of C4-C18ceramide, preferably C6 ceramide and/or C8 ceramide, included in theceramide anionic liposomes may be varied in proportion to the amount ofhydrophilic antineoplastic chemotherapeutic to be encapsulated in theceramide anionic liposomes such that the ratio of the C4-C18 ceramide,preferably C6 ceramide and/or C8 ceramide to hydrophilic antineoplasticchemotherapeutic is in the range of about 1:10-10:1.

Particular ratios of components included in liposomes according toaspects of the present invention are neutral lipid:modified neutrallipid:anionic lipid:C4-C18 ceramide—3-5.5 neutral lipid:1-4 modifiedneutral lipid:1 anionic lipid:3 C4-C18 ceramide.

Particular ratios of components included in liposomes according toaspects of the present invention are neutral lipid:modified neutrallipid:anionic lipid:C4-C18 ceramide—3:4:1:3.

Particular ratios of components included in liposomes according toaspects of the present invention are neutral lipid:modified neutrallipid:anionic lipid:C4-C18 ceramide—4.5:2:1:3.

Particular ratios of components included in liposomes according toaspects of the present invention are neutral lipid:modified neutrallipid:anionic lipid:C4-C18 ceramide—4.5:1.5:1:3.

Thus, according to preferred aspects, ceramide anionic liposomecompositions of the present invention include at least one modifiedneutral lipid, wherein the total amount of modified neutral lipid is anamount in the range of about 5-20 Molar percent, inclusive, morepreferably in the range of about 8-12 Molar percent, still morepreferably in the range of about 9-11 Molar percent and yet morepreferably about 10 Molar percent, wherein at least half of the amountof included modified neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of about 5-20 Molar percent,inclusive, more preferably in the range of about 8-12 Molar percent,still more preferably in the range of about 9-11 Molar percent and yetmore preferably about 10 Molar percent; C4-C18 ceramide in an amount inthe range of 1-40 Molar percent, inclusive, more preferably in the rangeof about 5-38 Molar percent, inclusive, still more preferably in therange of about 10-35 Molar percent, inclusive, more preferably 30 Molarpercent; and further including cationic or neutral lipids, with theproviso that the resulting ceramide anionic liposome compositions have anet negative charge at physiological pH.

Thus, according to highly preferred aspects, ceramide anionic liposomecompositions of the present invention include at least one pegylatedneutral lipid, wherein the total amount of pegylated neutral lipid is anamount in the range of about 5-20 Molar percent, inclusive, morepreferably in the range of about 8-12 Molar percent, still morepreferably in the range of about 9-11 Molar percent and yet morepreferably about 10 Molar percent, wherein at least half of the amountof included modified neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of about 5-20 Molar percent,inclusive, more preferably in the range of about 8-12 Molar percent,still more preferably in the range of about 9-11 Molar percent and yetmore preferably about 10 Molar percent; C4-C18 ceramide in an amount inthe range of 1-40 Molar percent, inclusive, more preferably in the rangeof about 5-38 Molar percent, inclusive, still more preferably in therange of about 10-35 Molar percent, inclusive, more preferably 30 Molarpercent; and further including cationic or neutral lipids, with theproviso that the resulting ceramide anionic liposome compositions have anet negative charge at physiological pH.

The term “Molar percent” as used herein to refer to amounts of C4-C18ceramide and neutral, anionic, cationic and modified lipids included inceramide anionic liposome compositions of the present invention, refersto the amount of the particular component as a Molar percent of totallipids in the ceramide anionic liposome compositions, excluding anyantineoplastic chemotherapeutic contained in the liposomes.

Ceramide anionic liposome compositions containing one or morehydrophilic antineoplastic chemotherapeutics are provided according toaspects of the present invention.

Ceramide anionic liposome compositions containing one or more vincaalkyloids are provided according to aspects of the present invention.

Ceramide anionic liposome compositions containing vinblastine,vincristine, vinglycinate, vinorelbine, vindesine, cladribine,sorafenib; or a combination of any two or more thereof are providedaccording to aspects of the present invention.

In addition to containing one or more hydrophilic antineoplasticchemotherapeutics and/or one or more vinca alkyloids, ceramide anionicliposome compositions of the present invention optionally contain any ofa variety of useful biologically active molecules and substancesincluding, but not limited to, proteins, peptides, carbohydrates,oligosaccharides, drugs, and nucleic acids capable of being complexedwith the liposomes. The term “biologically active molecules andsubstances” refers molecules or substances that exert a biologicaleffect in vitro and/or in vivo, such as, but not limited to, nucleicacids, inhibitory RNA, siRNA, shRNA, ribozymes, antisense nucleic acids,antibodies, hormones, small molecules, aptamers, decoy molecules andtoxins.

Methods and compositions are provided according to the present inventionfor treating cancer.

Methods of treatment of a subject having, or at risk of having, cancer,are provided according to aspects of the present invention includingadministration of a pharmaceutically effective amount of a ceramideanionic liposome composition containing one or more antineoplasticchemotherapeutics.

Methods of treatment of a subject having, or at risk of having, cancer,are provided according to aspects of the present invention includingadministration of a pharmaceutically effective amount of a ceramideanionic liposome composition containing one or more vinca alkyloids

Methods of treatment of a subject having, or at risk of having, cancer,are provided according to aspects of the present invention includingadministration of a pharmaceutically effective amount of a ceramideanionic liposome composition containing vinblastine, vincristine,vinglycinate, vinorelbine, vindesine, cladribine, sorafenib; or acombination of any two or more thereof.

Particular cancers treated using methods and compositions of the presentinvention are characterized by abnormal cell proliferation including,but not limited to, pre-neoplastic hyperproliferation, cancer in-situ,neoplasms and metastasis. Cancers treated using methods and compositionsof the present invention include solid tumors including, but not limitedto, cancers of the head and neck, esophagus, rectum, anus, prostate,testicle, lung, pancreas, bladder, ovary, uterus, cervix, thyroid,breast, colon, kidney, liver, brain and skin, as well as non-solidtumors, including, but not limited to, hematological malignancies suchas leukemia, lymphoma and multiple myeloma. Methods and compositions ofthe present invention can be used for prophylaxis as well asamelioration of signs and/or symptoms of cancer.

The terms “treating” and “treatment” used to refer to treatment of acancer in a subject include: preventing, inhibiting or ameliorating thecancer in the subject, such as slowing progression of the cancer and/orreducing or ameliorating a sign or symptom of the cancer.

A therapeutically effective amount of a composition of the presentinvention is an amount which has a beneficial effect in a subject beingtreated. In subjects having cancer or at risk for having cancer, such asa condition characterized by abnormal cell proliferation including, butnot limited to, pre-neoplastic hyperproliferation, cancer in-situ,neoplasms, metastasis, a tumor, a benign growth or other conditionresponsive to a composition of the present invention, a therapeuticallyeffective amount of a composition of the present invention is effectiveto ameliorate or prevent one or more signs and/or symptoms of thecondition. For example, a therapeutically effective amount of acomposition of the present invention is effective to detectably increaseapoptosis and/or decrease proliferation of cells of a cancer conditioncharacterized by abnormal cell proliferation including, but not limitedto, pre-neoplastic hyperproliferation, cancer in-situ, neoplasms,metastasis, a tumor, a benign growth or other condition responsive to acomposition of the present invention.

A subject treated according to methods and using compositions of thepresent invention can be mammalian or non-mammalian. A mammalian subjectcan be any mammal including, but not limited to, a human; a non-humanprimate; a rodent such as a mouse, rat, or guinea pig; a domesticatedpet such as a cat or dog; a horse, cow, pig, sheep, goat, or rabbit. Anon-mammalian subject can be any non-mammal including, but not limitedto, a bird such as a duck, goose, chicken, or turkey. In aspects ofmethods including administration of an inventive pharmaceuticalcomposition to a subject, the subject is human.

Optionally, methods of the present invention additionally includeadministration of one or more adjunct pharmacologically active agents.

Non-limiting examples of adjunct pharmacologically active agents thatcan be administered according to aspects of methods of the presentinvention include non-steroidal anti-inflammatory agents, antibiotics,antivirals, analgesics, antipyretics, antidepressants, antipsychotics,anticancer agents, antihistamines, anti-osteoporosis agents,anti-osteonecrosis agents, antiinflammatory agents, anxiolytics,chemotherapeutic agents, diuretics, growth factors, hormones and vasoactive agents.

Optionally, a method of treating a subject having cancer or at risk ofhaving cancer further includes an adjunct anti-cancer treatment. Anadjunct anti-cancer treatment can be administration of one or moreadditional antineoplastic chemotherapeutic agents, included or notincluded in inventive liposomes, administered separately or together.

A therapeutically effective amount of a pharmaceutical composition ofceramide anionic liposomes encapsulating a hydrophilic antineoplasticchemotherapeutic according to the present invention will vary dependingon the particular pharmaceutical composition used, the severity of thecondition to be treated, the species of the subject, the age and sex ofthe subject and the general physical characteristics of the subject tobe treated. One of skill in the art could determine a therapeuticallyeffective amount in view of these and other considerations typical inmedical practice. In general it is contemplated that a therapeuticallyeffective amount would be in the range of about 0.001 mg/kg-100 mg/kgbody weight, optionally in the range of about 0.01-10 mg/kg, and furtheroptionally in the range of about 0.1-5 mg/kg. Further, dosage may beadjusted depending on whether treatment is to be acute or continuing.

Antineoplastic chemotherapeutics are described, for example, in Goodmanet al., Goodman and Gilman's The Pharmacological Basis of Therapeutics,8th Ed., Macmillan. Publishing Co., 1990.

Antineoplastic chemotherapeutics illustratively include acivicin,aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin,allopurinol, altretamine, ambomycin, ametantrone, amifostine,aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenictrioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin,batimastat, benzodepa, bicalutamide, bisantrene, bisnafide dimesylate,bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin,calusterone, capecitabine, caracemide, carbetimer, carboplatin,carmustine, carubicin, carzelesin, cedefingol, celecoxib, chlorambucil,cirolemycin, cisplatin, cladribine, crisnatol mesylate,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate,diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone,duazomycin, edatrexate, eflornithine, elsamitrucin, enloplatin,enpromate, epipropidine, epirubicin, erbulozole, esorubicin,estramustine, etanidazole, etoposide, etoprine, fadrozole, fazarabine,fenretinide, floxuridine, fludarabine, fluorouracil, fluorocitabine,fosquidone, fostriecin, fulvestrant, gemcitabine, hydroxyurea,idarubicin, ifosfamide, ilmofosine, interleukin II (IL-2, includingrecombinant interleukin II or rIL2), interferon alfa-2a, interferonalfa-2b, interferon alfa-n1, interferon alfa-n3, interferon beta-Ia,interferon gamma-Ib, iproplatin, irinotecan, lanreotide, letrozole,leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol,maytansine, mechlorethamine hydrochloride, megestrol, melengestrolacetate, melphalan, menogaril, mercaptopurine, methotrexate, metoprine,meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin,mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid,nelarabine, nocodazole, nogalamycin, ormnaplatin, oxisuran, paclitaxel,pegaspargase, peliomycin, pentamustine, peplomycin, perfosfamide,pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin,plomestane, porfimer, porfiromycin, prednimustine, procarbazine,puromycin, pyrazofurin, riboprine, rogletimide, safingol, semustine,simtrazene, sorafenib, sparfosate, sparsomycin, spirogermanium,spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur,talisomycin, tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin,teniposide, teroxirone, testolactone, thiamiprine, thioguanine,thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestolone,triciribine, trimetrexate, triptorelin, tubulozole, uracil mustard,uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate,vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine,vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate,and zorubicin.

An adjunct anti-cancer treatment can be a radiation treatment of asubject or an affected area of a subject's body.

Hydrophilic antineoplastic chemotherapeutics are well-known in the art,including, but not limited to, vinblastine, vincristine, vinglycinate,vinorelbine, vindesine, sorafenib and cladribine.

Vinblastine is a well-known antineoplastic chemotherapeutic currentlyused in treatment of non-Hodgkin's lymphoma, small cell lung cancer,head and neck cancer, testicular cancers, breast cancers and variousgerm-cell cancers and compositions of the present invention may be usedto treat these and other cancers. Vincristine is a well-knownantineoplastic chemotherapeutic currently used in treatment of acutelymphocytic leukemias, multiple myelomas, rhabdomyosarcomas,neuroblastomas, Ewings sarcoma and Wilm's tumor and compositions of thepresent invention may be used to treat these and other cancers.Cladribine is a well-known antineoplastic chemotherapeutic currentlyused to treat hairy cell leukemias and compositions of the presentinvention may be used to treat these and other cancers. Sorafenib is awell-known antineoplastic chemotherapeutic currently used to treat renalcell carcinomas and hepatocellular carcinomas and compositions of thepresent invention may be used to treat these and other cancers.Vinorelbine is a well-known antineoplastic chemotherapeutic currentlyused to treat breast cancers and non-small cell lung cancers andcompositions of the present invention may be used to treat these andother cancers. Vindesine is a well-known antineoplastic chemotherapeuticcurrently used to treat leukemias, lymphomas, melanomas, breast cancersand lung cancers and compositions of the present invention may be usedto treat these and other cancers. Vinglycinate is a well-knownantineoplastic chemotherapeutic currently used in treatment of Hodgkin'sdisease, lymphosarcomas, lung cancers and chondrosarcomas andcompositions of the present invention may be used to treat these andother cancers.

Methods of the present invention include administration of ceramideanionic liposomes encapsulating one or more hydrophilic antineoplasticchemotherapeutics as pharmaceutical formulations, including thosesuitable for oral, rectal, nasal, pulmonary, epidural, ocular, otic,intraarterial, intracardiac, intracerebroventricular, intradermal,intravenous, intramuscular, intraperitoneal, intraosseous, intrathecal,intravesical, subcutaneous, topical, transdermal, and transmucosal, suchas by sublingual, buccal, vaginal, and inhalational, routes ofadministration.

Compositions including a hydrophilic antineoplastic chemotherapeuticencapsulated in ceramide anionic liposomes according to the presentinvention may be administered directly or may be formulated with one ormore additional pharmaceutically acceptable carriers where desired. Theterm “pharmaceutically acceptable carrier” refers to a carrier which issubstantially non-toxic to a subject to which the composition isadministered and which is substantially chemically inert with respect toceramide anionic liposomes and the encapsulated antineoplastictherapeutic of the present invention. Pharmaceutically acceptablecarriers and formulation of pharmaceutical compositions are known in theart, illustratively including, but not limited to, as described inRemington: The Science and Practice of Pharmacy, 21st Ed., Lippincott,Williams & Wilkins, Philadelphia, Pa., 2006; and Allen, L. V. et al.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed.,Lippincott, Williams & Wilkins, Philadelphia, Pa., 2005.

Pharmaceutical compositions suitable for delivery to a subject may beprepared in various forms illustratively including physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers include water, ethanol, polyols such aspropylene glycol, polyethylene glycol, glycerol, and the like, suitablemixtures thereof; vegetable oils such as olive oil; and injectableorganic esters such as ethyloleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants, such as sodium lauryl sulfate. Additionalcomponents illustratively including a buffer, a solvent, or a diluentmay be included.

Such formulations are administered by a suitable route includingparenteral and oral administration. Administration may include systemicor local injection, and particularly intravenous injection.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride, and substances similar in nature.Prolonged delivery of an injectable pharmaceutical form can be broughtabout by the use of agents delaying absorption, for example, aluminummonostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, at least oneinert customary excipient (or carrier) can be included such as sodiumcitrate or dicalcium phosphate or (a) fillers or extenders, as forexample, starches, lactose, sucrose, glucose, mannitol, and silicicacid, (b) binders, as for example, carboxymethylcellulose, alignates,gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, asfor example, glycerol, (d) disintegrating agents, as for example,agar-agar, calcium carbonate, plant starches such as potato or tapiocastarch, alginic acid, certain complex silicates, and sodium carbonate,(e) solution retarders, as for example, paraffin, (f) absorptionaccelerators, as for example, quaternary ammonium compounds, (g) wettingagents, as for example, cetyl alcohol, glycerol monostearate, andglycols (h) adsorbents, as for example, kaolin and bentonite, and (i)lubricants, as for example, talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.In the case of capsules, tablets, and pills, the dosage forms may alsoinclude a buffering agent.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include a pharmaceuticallyacceptable carrier formulated as an emulsion, solution, suspension,syrup, or elixir. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to a hydrophilic antineoplasticchemotherapeutic encapsulated in ceramide anionic liposomes, may containsuspending agents, as for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitol esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar or tragacanth,or mixtures of these substances, and the like.

A topical formulation can be an ointment, lotion, cream or gel inparticular aspects. Topical dosage forms such as ointment, lotion, creamor gel bases are described in Remington: The Science and Practice ofPharmacy, 21st Ed., Lippincott Williams & Wilkins, 2006, p. 880-882 andp. 886-888; and in Allen, L. V. et al., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, 8th Ed., Lippincott Williams & Wilkins,2005, p. 277-297.

A pharmaceutical composition according to the invention generallyincludes about 0.1-99% ceramide anionic liposomes encapsulating ahydrophilic antineoplastic therapeutic. Combinations of two or morepopulations of ceramide anionic liposomes encapsulating differenthydrophilic antineoplastic therapeutics in a pharmaceutical compositionare also considered within the scope of the present invention.

Methods of producing ceramide anionic liposomes containing one or morehydrophilic antineoplastic chemotherapeutics encapsulated in theinterior space of the anionic liposomes are provided according to thepresent invention.

Methods of producing ceramide anionic liposomes containing one or morehydrophilic vinca alkyloid antineoplastic chemotherapeutics encapsulatedin the interior space of the anionic liposomes are provided according tothe present invention.

Methods of producing ceramide anionic liposomes containing one or moreantineoplastic chemotherapeutics containing one or more of: vinblastine,vincristine, vinglycinate, vinorelbine, vindesine, sorafenib andcladribine, encapsulated in the interior space of the ceramide anionicliposomes are provided according to the present invention.

Preparation of the ceramide anionic liposomes encapsulating one or morehydrophilic antineoplastic chemotherapeutics is performed with specialreference to temperatures used, in contrast to previous methods. Inparticular, an extrusion step is performed at a temperature in the rangeof 67-75 degrees Celsius, inclusive, more preferably in the range of67-69 degrees Celsius, inclusive, and most preferably at 68 degreesCelsius. The steps of rehydrating the lipid mixture in an aqueouspharmaceutically acceptable liquid characterized by physiological pH,and the first and second sonication steps are performed at a temperaturein the range of 55-75 degrees Celsius, inclusive, more preferably 60-70degrees Celsius, inclusive, and most preferably at 65 degrees Celsius.This “trapping” mechanism of encapsulation that uses temperature insteadof traditional pH-based methodologies to achieve formation ofpharmaceutical formulations of ceramide anionic liposomes containing atherapeutic dose of vinca alkyloids and other hydrophilic antineoplasticchemotherapeutics, avoiding premature degradation of the vinca alkyloidsand other hydrophilic antineoplastic chemotherapeutics.

The phrase “aqueous pharmaceutically acceptable liquid” with referenceto “rehydration” of a lipid mixture according to aspects of the presentinvention refers to a liquid which is substantially non-toxic to asubject and which is substantially chemically inert with respect to thelipids and the antineoplastic chemotherapeutic to be encapsulated. Anon-limiting example is phosphate buffered saline/normal saline.

Methods of producing a pharmaceutical composition are provided accordingto aspects of the present invention which include providing a lipidmixture comprising: at least one pegylated neutral lipid, wherein thetotal amount of pegylated neutral lipid is an amount in the range of5-20 Molar percent, inclusive, of the total lipid mixture and wherein atleast half of the amount of included pegylated neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of 5-15 Molar percent,inclusive, of the total lipid mixture; C6 ceramide in an amount in therange of 1-40 Molar percent, inclusive, of the total lipid mixture; andcationic or neutral lipids, with the proviso that the resulting lipidmixture has a net negative charge at physiological pH. The lipid mixtureis dried to remove solvents under nitrogen and then suspended in anaqueous pharmaceutically acceptable liquid. A hydrophilic antineoplasticchemotherapeutic is added to the lipid mixture suspended in the aqueouspharmaceutically acceptable buffer such that the ratio of the amount ofthe hydrophilic antineoplastic chemotherapeutic to the amount of totallipids in the suspended lipid mixture is in the range of about1:1-1:100. The combination of lipids and drug is then sonicated at atemperature in the range of 55-75 degrees Celsius, inclusive, to producea sonicated mixture. The sonicated mixture is then passed through afilter having pores of a desired size to produce liposomes having thedesired size, at a temperature in the range of 67-75 degrees Celsius,producing a population of ceramide anionic liposomes, wherein thepopulation of ceramide anionic liposomes contains greater than 10%,greater than 25% or greater than 50% of the amount of the hydrophilicantineoplastic chemotherapeutic added to the lipid mixture, encapsulatedin the aqueous core of the liposomes.

Methods of producing a pharmaceutical composition are provided accordingto aspects of the present invention which include providing a lipidmixture comprising: at least one pegylated neutral lipid, wherein thetotal amount of pegylated neutral lipid is an amount in the range of5-20 Molar percent, inclusive, of the total lipid mixture and wherein atleast half of the amount of included pegylated neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of 5-15 Molar percent,inclusive, of the total lipid mixture; C6 ceramide in an amount in therange of 1-40 Molar percent, inclusive, of the total lipid mixture; andcationic or neutral lipids, with the proviso that the resulting lipidmixture has a net negative charge at physiological pH. The lipid mixtureis dried to remove solvents under nitrogen and then suspended in anaqueous pharmaceutically acceptable liquid. A hydrophilic antineoplasticchemotherapeutic is added to the lipid mixture suspended in the aqueouspharmaceutically acceptable buffer such that the ratio of the amount ofthe hydrophilic antineoplastic chemotherapeutic to the amount of totallipids in the suspended lipid mixture is in the range of about1:1-1:100. The combination of lipids and drug is then sonicated at atemperature in the range of 55-75 degrees Celsius, inclusive, to producea sonicated mixture. The sonicated mixture is then passed through afilter having pores of a desired size to produce liposomes having thedesired size, at a temperature in the range of 67-75 degrees Celsius,producing a population of ceramide anionic liposomes, wherein thepopulation of ceramide anionic liposomes contains greater 15-75% of theamount of the hydrophilic antineoplastic chemotherapeutic added to thelipid mixture, encapsulated in the aqueous core of the liposomes.

Commercial Packages

Commercial packages are provided according to aspects of the presentinvention for treating cancer in a subject in need thereof, includingone or more hydrophilic antineoplastic chemotherapeutics encapsulated inceramide anionic liposomes. One or more auxiliary components areoptionally included in commercial packages of the present invention,such as a pharmaceutically acceptable carrier exemplified by a buffer ordiluent.

Aspects of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES Example 1

Table I shows amounts of the indicated materials included in liposomesused in this example.

TABLE I MW Stock (mg/ Mg Molar (mg/ μl Lipid mmol) Lipid μmol Ratio ml)(1 ml) DSPC 790.16 9.0746 11.4845 3.75 25 362.98 DOPE 744.04 3.98765.3594 1.75 25 159.51 PEG(2000)- 2805.54 4.2960 1.5313 0.5 25 171.84DSPE PEG(750)- 1244.64 1.9059 1.5313 0.5 25 76.23 C8 Dihexadecyl 546.862.4138 4.4140 1 25 96.55 Phosphate C6-Cer 397.64 3.1160 7.8361 3 25124.64 Total x 24.7939 32.1565 10.5 25 991.76

Abbreviations for Table I: DSPC, distearoylphosphatidylcholine; DOPEdioleoylphosphatidylethanolamine; PEG(2000) DSPE, polyethyleneglycol2000 distearoylphosphatidylethanolamine; PEG(750) C8,N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750;C6-cer, C6 ceramide.

Methods according to aspects of the present invention for makinghydrophilic antineoplastic chemotherapeutic-containing ceramide anionicliposomes allow achievement of high encapsulation efficiencies with astable anionic nanoscale liposome formulation. The lipids to be includedin the liposomes are combined in a mixture, dried under nitrogen toremove solvents and then rehydrated in PBS/normal saline at 65 degreesCelsius for up to 3 hours, followed by sonication for two minutes. Inthis example, five milligrams vinblastine in 100 microliters ofphosphate buffered saline/0.9% NaCl is added to 900 microliters of thelipid mixture such that the vinblastine concentration is 5 mg/mL and thevinblastine:lipid ratio is 5 mg drug:24.79 mg lipid, followed byovernight incubation (12-18 hrs) at room temperature. After theovernight incubation, the mixture was warmed to 65 degrees Celsius for30 minutes, followed by a second sonication at 65 degrees Celsius for 5minutes until translucent and then extrusion at 68 degrees Celsius usinga 100 nM filter within a miniextruder (9 passages), producing a “finalproduct” of 90 nanometer sized ceramide anionic liposomes including 28.5molar percent short chain ceramide, 9.5 molar percent pegylated shelland 9.5 molar percent anionic dihexadecyl phosphate which allows forencapsulation in the void volume of the liposome of between 3 to 4 mg ofvinblastine from an initial concentration of 5 mg/ml. These values aredetermined by LC/MS methodologies and reveal no premature degradation ofthe vinblastine as a function of encapsulation. These encapsulationefficiencies of nearly 75% are the result of using the anionicformulations described herein. Without inclusion of anionic lipid inliposomes as described herein, efficiencies of encapsulation ofvinblastine are below 10% and the resulting liposomes are highlyunstable. These final products produced in this Example encapsulate atherapeutic molar dose of vinblastine of 4 mM within ceramide anionicliposomes containing 8.8 μM ceramide.

The final product was run through a CL-4B sepharose column to removefree vinblastine. Final products are stored at room temperature.

These nanoscale preparations of 90 nm sized liposomes are stable for atleast 2 months as evidenced by a lack of change in size (FIG. 1) andzeta potential charge measurements. Specifically, the dynamic lightscattering measurements do not change as a function of time (7, 14, 21days).

Biological evidence of combinatorial efficacy of the ceramide anionicanionic liposomes encapsulating vinblastine includes cellrespiration/survival data, in which a synergistic effect of thecombinatorial ceramide/vinblastine product is observed compared toeither agent alone in pancreatic tumor cells. Pane 1 pancreatic cancercells were treated with 3.2 μM anionic (A) and neutral ceramide (C6) ornon-ceramide nanoliposomes that either contained 0.01, 0.1 or 1 μMvinblastine or PBS. Ghost liposomes contained no ceramide orvinblastine. Unencapsulated (free) vinblastine was also administered atsimilar concentrations. All treatments were for 24 hrs.

Cell viability was assessed by MTS assay, which measures cellularrespiration as a function of reduction of MTS to formazene at 490 nm.Only the high concentration of free vinblastine reduced cell viability.Ceramide nanoliposomes had minimal affects upon cellular viability. Incontrast, the ceramide anionic/vinblastine nanoliposome had significantaffects upon cellular viability. One way ANOVA revealed a p<0.05statistical difference between free vinblastine and the encapsulatedvinblastine within a ceramide nanoliposome. N=6 individual samples foreach condition, replicated in triplicate.

FIG. 2 is a graph showing data from these experiments showing thatformulated C6 ceramide/vinblastine nanoliposomes are efficacious in atumor model as compared to either C6 ceramide nanoliposomes orvinblastine alone. Abbreviations used in FIG. 2: PBS: Phosphate bufferedSaline; Ghost: neutral liposome (no ceramide); AC6: Anionic C6 liposome;Vin: Vinblastine alone; C6: Neutral C6 ceramide; liposome A-Ghost:Anionic liposome (no ceramide); AC6+VIN: Ceramide anionic liposome thatcontains vinblastine.

Similar procedures are followed using hydrophilic antineoplasticchemotherapeutic drugs, such as sorafenib and cladribine, or other vincaalkyloids such as vincristine, vinglycinate, vinorelbine and vindesine,to obtain similar drug loading and anti-cancer effects.

Example 2

Table II shows amounts of the indicated materials included in liposomesused in this example.

TABLE II MW Stock (mg/ Mg Molar (mg/ μl Lipid mmol) Lipid μmol Ratio ml)(1 ml) DSPC 790.16 7.2212 9.1389 2.75 25 288.85 DOPE 744.04 4.32715.8156 1.75 25 173.08 PEG(2000)- 2805.54 6.9926 2.4924 0.75 25 279.70DSPE PEG(750)- 1244.64 3.1022 2.4924 0.75 25 124.09 C8 Dihexadecyl546.86 2.4138 4.4140 1 25 96.55 Phosphate C6-Cer 397.64 3.5306 8.8789 325 141.22 Total x 27.5874 33.2322 10 25 1103.50

Abbreviations for Table II: DSPC, distearoylphosphatidylcholine; DOPEdioleoylphosphatidylethanolamine; PEG(2000) DSPE, polyethyleneglycol2000 distearoylphosphatidylethanolamine; PEG(750) C8,N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750;C6-Cer, C6 ceramide.

Liposomes are made according to the procedure described in Example 1using the materials and amounts listed in Table II to produce ceramideanionic liposomes including one or more encapsulated hydrophilicantineoplastic chemotherapeutic drugs, such as sorafenib and cladribine,or other vinca alkyloids such as vincristine, vinglycinate, vinorelbineand vindesine, to obtain similar drug loading and anti-cancer effects.

Example 3

Table III shows amounts of the indicated materials included in liposomesused in this example.

TABLE III MW Stock (mg/ Mg Molar (mg/ μl Lipid mmol) Lipid μmol Ratioml) (1 ml) DSPC 790.16 6.9263 8.7657 2.75 25 277.05 DOPE 744.04 4.15045.5782 1.75 25 166.02 PEG(2000)- 2805.54 8.9427 3.1875 1 25 357.71 DSPEPEG(750)- 1244.64 3.9673 3.1875 1 25 158.69 C8 Dihexadecyl 546.86 2.41384.4140 1 25 96.55 Phosphate C6-Cer 397.64 3.3148 8.3361 3 25 132.59Total x 29.7154 33.4690 10.5 25 1188.61

Abbreviations for Table III: DSPC, distearoylphosphatidylcholine; DOPEdioleoylphosphatidylethanolamine; PEG(2000) DSPE, polyethyleneglycol2000 distearoylphosphatidylethanolamine; PEG(750) C8,N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750;C6-Cer, C6 ceramide.

Liposomes are made according to the procedure described in Example 1using the materials and amounts listed in Table III to produce ceramideanionic liposomes including one or more encapsulated hydrophilicantineoplastic chemotherapeutic drugs, such as sorafenib and cladribine,or other vinca alkyloids such as vincristine, vinglycinate, vinorelbineand vindesine, to obtain similar drug loading and anti-cancer effects.

Example 4

Table IV shows amounts of the indicated materials included in liposomesused in this example.

TABLE IV MW Stock (mg/ Mg Molar (mg/ μl Lipid mmol) Lipid μmol Ratio ml)(1 ml) DSPC 790.16 5.0373 6.3751 2 25 201.49 DOPE 744.04 2.3716 3.1875 125 94.87 PEG(2000)- 2805.54 17.8855 6.3751 2 25 715.42 DSPE PEG(750)-1244.64 7.9347 6.3751 2 25 317.39 C8 Dihexadecyl 546.86 2.4138 4.4140 125 96.55 Phosphate C6-Cer 397.64 3.3148 8.3361 3 25 132.59 Total x38.9577 35.0628 11 25 1558.31

Abbreviations for Table IV: DSPC, distearoylphosphatidylcholine; DOPEdioleoylphosphatidylethanolamine; PEG(2000) DSPE, polyethyleneglycol2000 distearoylphosphatidylethanolamine; PEG(750) C8,N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750;C6-Cer, C6 ceramide.

Liposomes are made according to the procedure described in Example 1using the materials and amounts listed in Table IV to produce ceramideanionic liposomes including one or more encapsulated hydrophilicantineoplastic chemotherapeutic drugs, such as sorafenib and cladribine,or other vinca alkyloids such as vincristine, vinglycinate, vinorelbineand vindesine, to obtain similar drug loading and anti-cancer effects.

Example 5

Normal and cancer cell viability in the presence or absence of apharmaceutical composition can be determined according to a well-knownMTS method.

MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium),in the presence of phenazine methosulfate (PMS), produces a reducedformazan product, whose purple color can be assessed. In living cells,reduced formazan has an absorbance maximum at 490-500 nm inphosphate-buffered saline. The MTS assay is often described as a‘one-step’ MTT assay, which offers the convenience of adding the reagentstraight to the cell culture without the intermittent steps required inthe MTT assay. However this convenience makes the MTS assay susceptibleto colormetric interference as the intermittent steps in the MTT assayremove traces of colored compounds, whilst these remain in themicrotiter plate in the one-step MTS assay.

Vinblastine potentiates the inhibitory activity of C6 ceramidenanoliposomes upon growth and survival of cultured primary human AML ofpoor prognosis (#441) in semisolid media for clonogeneic growth as shownin FIG. 3A.

For FIG. 3A, primary human AML cells (n=5) were thawed and grown for12-14 days in semi-solid medium with the indicated concentration ofLip-C6 or Lip-ghost and/or vinblastine. Ceramide inhibits colonyformation in a dose-dependent manner.

In vivo therapeutic activity of C6-VBL (“combo”) nanoliposomes preparedas described in Example 1 is tested against poor prognosis hAML #329cells growing in NSG mice. FIG. 3B shows the blood burden of human AML(in 1000 cells/ul of blood) and the effect of compound C6-VBL (“combo”)nanoliposomes prepared as described in Example 1 vs control PBS or ghostnanoliposomes (p-0.0007 and 0.0022 respectively).

FIG. 3C shows similar results demonstrated for marrow replacement byhAML 329. C6, Ghost, and PBS are all significantly different than C6-VBL(“combo”) nanoliposomes prepared as described in Example 1 (p=0.0009,0.0016, and 0.0015 respectively). Similar results may be obtained usingthe ceramide anionic nanoliposomes of Examples 2-4 and 6-10.

Example 6

Table V shows amounts of the indicated materials included in liposomesused in this example.

TABLE V MW Stock (mg/ Mg Molar (mg/ ul Lipid mmol) Lipid umol Ratio ml)(1 ml) DSPC 790.16 9.4450 11.9532 3.75 25 377.80 DOPE 744.04 4.15045.5782 1.75 25 166.02 PEG(2000)- 2805.54 4.4714 1.5938 0.5 25 178.85DSPE PEG(750)- 1244.64 1.9837 1.5938 0.5 25 79.35 C8 Dihexadecyl 546.862.4138 4.4140 1 25 96.55 Phosphate C8-Cer 425.7 3.5487 8.3361 3 25141.95 Total x 26.0129 33.4690 10.5 25 1040.52

Abbreviations for Table IV: DSPC, distearoylphosphatidylcholine; DOPEdioleoylphosphatidylethanolamine; PEG(2000) DSPE, polyethyleneglycol2000 distearoylphosphatidylethanolamine; PEG(750) C8,N-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750;C8-Cer, C8 ceramide.

Liposomes are made according to the procedure described in Example 1using the materials and amounts listed in Table V to produce ceramideanionic liposomes including one or more encapsulated hydrophilicantineoplastic chemotherapeutic drugs, such as sorafenib and cladribine,or other vinca alkyloids such as vincristine, vinglycinate, vinorelbineand vindesine, to obtain similar drug loading and anti-cancer effects.

Example 7

In this example, the lipids listed in Table II are combined in amixture, dried under nitrogen to remove solvents and then rehydrated inphosphate buffered saline, pH 7.4, at 65 degrees Celsius for up to 3hours, followed by sonication for two minutes. Five milligramsvincristine in 100 microliters of phosphate buffered saline/0.9% NaCl isadded to 900 microliters of the lipid mixture such that the drugconcentration is 5 mg/mL and the drug:lipid ratio is 5 mg drug:27.59 mglipid, followed by overnight incubation (12-18 hrs) at room temperature.After the overnight incubation, the mixture is warmed to 65 degreesCelsius for 30 minutes, followed by a second sonication at 65 degreesCelsius for 5 minutes until translucent and then extrusion at 68 degreesCelsius using a 100 nM filter within a miniextruder (9 passages), toproduce a “final product” of ceramide anionic liposomes encapsulating 3to 4 mg of vincristine.

To demonstrate anti-cancer activity, DLD1 human colon cancer cells aretreated with ceramide anionic liposomes that contain 0.01 μMvincristine, 0.1 μM vincristine, 1 μM vincristine, PBS or varyingamounts of the ceramide anionic liposomes of Example 1. Controls can beghost liposomes containing no ceramide, vincristine or vinblastine; orunencapsulated (free) vinblastine or vincristine at similarconcentrations.

Example 8

In this example, the lipids listed in Table V are combined in a mixture,dried under nitrogen to remove solvents and then rehydrated in phosphatebuffered saline, pH 7.4, at 65 degrees Celsius for up to 3 hours,followed by sonication for two minutes. Five milligrams vinblastine in100 microliters of phosphate buffered saline/0.9% NaCl is added to 900microliters of the lipid mixture such that the drug concentration is 5mg/mL and the drug:lipid ratio is 5 mg drug:26.01 mg lipid, followed byovernight incubation (12-18 hrs) at room temperature. After theovernight incubation, the mixture is warmed to 65 degrees Celsius for 30minutes, followed by a second sonication at 65 degrees Celsius for 5minutes until translucent and then extrusion at 68 degrees Celsius usinga 100 nM filter within a miniextruder (9 passages), to produce a “finalproduct” of ceramide anionic liposomes encapsulating 3 to 4 mg ofvinblastine.

To demonstrate anti-cancer activity, DLD1 human colon cancer cells aretreated with ceramide anionic liposomes that contain 0.01 μMvinblastine, 0.1 μM vinblastine, 1 μM vinblastine or PBS. Controls canbe ghost liposomes containing no ceramide or vinblastine; orunencapsulated (free) vinblastine at similar concentrations.

Example 9

In this example, the lipids listed in Table II are combined in amixture, dried under nitrogen to remove solvents and then rehydrated inphosphate buffered saline, pH 7.4, at 65 degrees Celsius for up to 3hours, followed by sonication for two minutes. Five milligramscladribine in 100 microliters of phosphate buffered saline/0.9% NaCl isadded to 900 microliters of the lipid mixture such that the drugconcentration is 5 mg/mL and the drug:lipid ratio is 5 mg drug:27.59 mglipid, followed by overnight incubation (12-18 hrs) at room temperature.After the overnight incubation, the mixture is warmed to 65 degreesCelsius for 30 minutes, followed by a second sonication at 65 degreesCelsius for 5 minutes until translucent and then extrusion at 68 degreesCelsius using a 100 nM filter within a miniextruder (9 passages), toproduce a “final product” of ceramide anionic liposomes encapsulating 3to 4 mg of cladribine.

To demonstrate anti-cancer activity, MDA-MB-468 human breast cancercells are treated with ceramide anionic liposomes that contain 0.01 μMcladribine, 0.1 μM cladribine, 1 μM cladribine, PBS or varying amountsof the ceramide anionic liposomes of Example 1. Controls can be ghostliposomes containing no ceramide, cladribine or vinblastine; orunencapsulated (free) vinblastine or cladribine at similarconcentrations.

Example 10

In this example, the lipids listed in Table I are combined in a mixture,dried under nitrogen to remove solvents and then rehydrated in phosphatebuffered saline, pH 7.4, at 65 degrees Celsius for up to 3 hours,followed by sonication for two minutes. Five milligrams sorafenib in 100microliters of phosphate buffered saline/0.9% NaCl is added to 900microliters of the lipid mixture such that the drug concentration is 5mg/mL and the drug:lipid ratio is 5 mg drug:27.59 mg lipid, followed byovernight incubation (12-18 hrs) at room temperature. After theovernight incubation, the mixture is warmed to 65 degrees Celsius for 30minutes, followed by a second sonication at 65 degrees Celsius for 5minutes until translucent and then extrusion at 68 degrees Celsius usinga 100 nM filter within a miniextruder (9 passages), to produce a “finalproduct” of ceramide anionic liposomes encapsulating 3 to 4 mg ofsorafenib.

To demonstrate anti-cancer activity, SK-HEP-1human hepatocellular cancercells and UACC-903 human melanoma cells are treated with ceramideanionic liposomes that contain 0.01 sorafenib, 0.1 μM sorafenib, 1 μMsorafenib, PBS or varying amounts of the ceramide anionic liposomes ofExample 1. Controls can be ghost liposomes containing no ceramide,sorafenib or vinblastine; or unencapsulated (free) vinblastine orsorafenib at similar concentrations.

Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference.

The compositions and methods described herein are presentlyrepresentative of preferred aspects, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

1. A pharmaceutical composition, comprising: a antineoplasticchemotherapeutic encapsulated in ceramide anionic liposomes.
 2. Thepharmaceutical composition of claim 1, wherein the antineoplasticchemotherapeutic is an antineoplastic vinca alkyloid.
 3. Thepharmaceutical composition of claim 2, wherein the antineoplasticchemotherapeutic is selected from the group consisting of: vinblastine,vincristine, vinglycinate, vinorelbine, vindesine, sorafenib, cladribineand a combination of any two or more thereof.
 4. The pharmaceuticalcomposition of claim 1, wherein the ceramide anionic liposomes comprisea 3-5.5:1-4:1:3 ratio of neutral lipid:pegylated neutral lipid:anioniclipid:C4-C18 ceramide.
 5. The pharmaceutical composition of claim 4,wherein the neutral lipid is a mixture of DSPC and DOPE, wherein thepegylated neutral lipid is a mixture of PEG(2000)-DSPE andN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750, andwherein the anionic lipid is dihexadecyl phosphate.
 6. Thepharmaceutical composition of claim 1, wherein the antineoplasticchemotherapeutic is vinblastine.
 7. The pharmaceutical composition ofclaim 1, wherein the liposomes comprise at least one pegylated neutrallipid, wherein the total amount of pegylated neutral lipid is an amountin the range of 5-20 Molar percent, inclusive, and wherein at least halfof the amount of included modified neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of 5-15 Molar percent,inclusive; one or more ceramides selected from C4 ceramide, C6 ceramide,C8 ceramide, C10 ceramide, C12 ceramide, C14 ceramide, C16 ceramide andC18 ceramide, in an amount in the range of 1-40 Molar percent,inclusive; and cationic or neutral lipids, with the proviso that theresulting ceramide anionic liposomes have a net negative charge atphysiological pH.
 8. A method of treatment of a subject in need thereof,comprising: administration of a therapeutically effective amount of thecomposition of claim
 1. 9. The method of claim 8, wherein the subjecthas, or is at risk of having, cancer.
 10. The method of claim 8, furthercomprising administration of an adjunct anti-cancer treatment.
 11. Amethod of producing a pharmaceutical composition, comprising: providinga lipid mixture comprising at least one pegylated neutral lipid, whereinthe total amount of pegylated neutral lipid is an amount in the range of5-20 Molar percent, inclusive, and wherein at least half of the amountof included pegylated neutral lipid isN-Octanoyl-Sphingosine-1-succinyl(methoxy(polyethylene)glycol)750(PEG(750) C8); at least one anionic lipid, wherein the total amount ofanionic lipid is an amount in the range of 5-15 Molar percent,inclusive; one or more ceramides selected from C4 ceramide, C6 ceramide,C8 ceramide, C10 ceramide, C12 ceramide, C14 ceramide, C16 ceramide andC18 ceramide, in an amount in the range of 1-40 Molar percent,inclusive; and cationic or neutral lipids, with the proviso that theresulting lipid mixture has a net negative charge at physiological pH;sonicating the lipid mixture in the presence of an amount of aantineoplastic chemotherapeutic at a temperature in the range of 55-75degrees Celsius, inclusive, to produce a sonicated mixture; and passingthe sonicated mixture through a filter having pores of a desired size toproduce liposomes having the desired size, at a temperature in the rangeof 67-75 degrees Celsius, producing a population of ceramide anionicliposomes, wherein the population comprises greater than 10% of theamount of the antineoplastic chemotherapeutic.
 12. The method of claim11, wherein the population comprises greater than 25% of the amount ofthe antineoplastic chemotherapeutic.
 13. The method of claim 11, whereinthe population comprises 15-75% of the amount of the antineoplasticchemotherapeutic.
 14. The method of claim 11, wherein the ceramide is C6ceramide, C8 ceramide or a combination of C6 ceramide and C8 ceramide.15. The method of claim 11, wherein the antineoplastic chemotherapeuticis a vinca alkyloid antineoplastic chemotherapeutic.
 16. The method ofclaim 11, wherein the antineoplastic chemotherapeutic is selected fromthe group consisting of: vinblastine, vincristine, vinglycinate,vinorelbine, vindesine, sorafenib, cladribine and a combination of anytwo or more thereof.
 17. The method of claim 11, wherein theantineoplastic chemotherapeutic is vinblastine.